-
Isolation, Partial Characterization and Modification of the
Great Northern Bean (Phaseolus vulgaris L.) Starch
S. K. SATHE and D. K. SALUNKHE
ABSTRACT Microscopy of starch The yield of the Great Northern
bean starch was 18.23% (bean flour basis). The starch granule size
ranged from 12 x 12 pm to 58 x 40 pm (length x width). The shape of
starch granules was round to oval to elliptical, and in some cases,
concave as well. Lamellae were pres- ent on all the starch granules
observed. Amylose content of the starch was 10.2% (starch basis).
Hog pancreatic cY-amylase hydrolyzed more starch than did malt
a-amylase under similar conditions. The Great Northern bean starch
had good water and oil absorption capa- cities at room temperature
(21C). The bean starch formed a stable gel at concentrations of 7%
and above (w/v). The viscoamylographic studies of the isolated
starch indicated the restricted-swelling char- acter of the bean
starch.
The purified starch was studied microscopically by employing
both light and scanning electron microscope. For light micro-
scopic studies, starch was moistened with a drop of distilled
water. A calibrated eyepiece lens (152.5@ (calibrated against a
stage micrometer) was employed for the measurements of starch
granule size at a magnification of 344. The starch granules were
studied for the size, shape, hylum, and lamellae. The size
measurements were made on 25 representative granules.
INTRODUCTION
Starch samples for scanning electron microscopic studies were
prepared as follows. Starch was sprinkled on an aluminum stub (with
a double-stick tape on it) and was coated with a gold-palla- dium
alloy completely in a Polaron E 5000 Sputter Coater (U.K.) and the
specimens observed in AMR 1OOOB scanning electron micro- scope
(Cambridge, Mass.) at 20 KV accelerating voltage and suitable
magnification(s).
LEGUMES contain about 60% carbohydrates including starch,
reducing and nonreducing sugars, oligosaccharides of the raffinose
family, and others. Starch constitutes the major portion of legume
carbohydrates. Cerning-Beroard and Filiatre (1976) studied the
carbohydrate composition of horsebeans, smooth and wrinkled peas,
and lupine seeds. They found that the average starch content of
horsebeans, smooth and wrinkled peas, and lupine seeds to be 41 .O,
48.0, 33.0, and 0.4%, respectively. Naivikul (1977) re- ported the
starch content range to be 50.9-52.9% (mois- ture free basis) in
navy bean, pinto bean, faba bean, lentil, and mung bean. Schoch and
Maywald (1968) discussed the difficulties encountered in the
separation of horsebean starch. They attributed these difficulties
to the presence of a highly hydrated fine fiber fraction
(presumably from the cell walls enclosing the starch granules) and
high content of insoluble proteins. Halbrook and Kurtzman (1975)
studied the water uptake by the Great Northern bean starch at high
(80-148(Z) temperatures. Recently, Lai and Varriano- Marston (1979)
reported certain physicochemical charac- teristics of black bean
starch.
Hydrolysis of starch Starch was hydrolysed by two different
ol-amylases (from Hog
pancreas, type VI-A, and from malt, type V-A; both from
Sigma
I RESIDUE F 30L 2% N&I, 24 h, 4C
1 RESIDUE 1 1 Washed with 2L H,O, blended with 6L O.IN NaOH (1
min.1 m a Warmg Blendor and extracted for 48 h. 4C
I CENTRIFUGE I- 10,000 RPM, 30 min. The purpose of the present
investigation was to isolate
the Great Northern bean starch and to study certain physi-
cochemical properties of the unmodified and modified starch.
MATERIALS & METHODS The Great Northern beans were purchased
from Bean Growers Warehouse, Filer, Idaho, and stored at 4C until
experiments were conducted. The residue obtained after preparation
of the protein concentrates on a pilot plant scale (Sathe et al.,
1980) was referred to as crude starch. All the chemicals were of
reagent grade unless mentioned otherwise. All the analyses were
performed in triplicate and means reported.
I RESUSPEND IN 80% AQ. ETHANOL 4 * 4
HEATING WATER BATH
Isolation of starch The beans were ground to 20 mesh in a Fitz
mill (The W.J.
Fitzpatric Co., Chicago, Ill.). Three kg of bean flour were
extracted sequentially with different solvents to yield starch. The
schematic diagram for the process is presented in Figure 1.
FREEZE DEHYDRATE
1 STARCH POWDER I
Authors Sathe and Salunkhe are affiliated with the Dept. of
Nutri- tion & Food Sciences, Utah State Univ., Logan, UT84322.
Fig. l-Schematic diagram for the isolation of Great Northern
bean
starch.
L Blended 1 min. in a Waring Blendor 80C. 1 h
4h. 4-C; Discard supernatant
Volume 46 (1981kJOURNAL OF FOOD SCIENCE-617
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Chemical Co., St. Louis, MO.) according to the method described
by Decker (1977). Maltose hydrate (Grade II, Sigma Chemical Co.,
St. Louis, MO.) served as reference standard. The starch to enzyme
ratio in each case was 1:l (w/w). Liberated maltose was measured,
after inactivating the enzyme (heating in a boiling water bath for
3 min), calorimetrically (3,5dinitrosalicylic acid as color
reagent) at 540 nm in a Beccman-DBG spectrophotometer. Maltose
equiva- lent was determinec at time intervals of 0, 15, 30, 60,90,
and 120 min. The incubatiorI was, in both cases, at pH 7.0 and at a
tempera- ture of 21C.
Modifications Acetylation-The method of Wurzburg (1964) was
followed.
One hundred grams of starch were dispersed in 500 ml of
distilled water and magnetic:ally stirred for 30 min to obtain a
uniform suspension. The pH 1 rf the slurry was adjusted to 8.0 with
1N NaOH. Acetic anhydride (11.2g) was then added slowly to this
slurry main- taining constant stirring and monitoring the pH
between 8.0-8.4. The reaction was al.owed to proceed for an
additional 5 mm after completion of the acetic anhydride addition.
The pH of the slurry was finally adjusted o 4.5 with 0.5N HCl and
filtered through What- man filter paper #4. The residue was then
washed five times with distilled water and freeze dehydrated.
Oxidation-A uniform slurry of starch (1OOg starch in 500 ml
distilled water) was prepared as in acetylation. Oxidation of the
purified starch wa:. accomplished by the method of Hullinger
(1964). The pH of the slurry was first adjusted to 9.0-9.5 with 3%
aqueous NaOH and log of NaOCl was added slowly (over a period of 90
min) while rraintaining the magnetic stirring and constantly
monitoring the pH lletween 9.0-9.5. Cooling was provided (crushed
ice with NaCl) simitltaneously. The reaction was allowed to pro-
ceed for 4 hr after IJaOCl addition was completed, pH adjusted to
7.0 with 0.5N HCI and the slurry was filtered through Whatman
filter paper #4. Th: residue was washed five times with distilled
water and freeze dehydrated.
Moisture The moisture content of the samples were determined by
the
AACC method 44-15 (1962).
Proteins Protein content of the appropriate .samples was
determined
by the Kjeldahl method (N X 6.25).
Fat The method followed was that of Schoch (1964). Starch
(5g)
was hydrolyzed witlr 8N HCl solution for 1 hr on a boiling water
bath. After successb.e extraction of fat with ether and petroleum
ether, the solvents Mere evaporated on a hot water bath (80C) and
the nearly dry samples were then dried in an oven (100C) for 20 min
followel by three successive extractions with carbon tetrachloride
(10 m. each time). The combined extracts were fil- tered and the
solvc,nt evaporated on a hot water bath (SOC). The beakers (previot
sly weighed) containing the nearly dry samples were dried in an
oven (100C) for 30 mm and weighed. The differ- ence was interpretet
as the weight of fat. The fat content was re- ported on a dry
weigrt basis.
Amylose content Amylose content of the purified starch was
determined by the
procedure of Wolf et al. (1970) with slight modifications. Pure
amy- lose (Potato, Type 111, Sigma Chemical Co., St. Louis, MO.)
served as standard. Starch lvas dissolved in 90% (v/v) dimethyl
sulfoxide and 0.2, 0.5, and 1.(1 ml portions were assayed for the
amylose con- tent. The starch concentration in 90% dimethyl
sulfoxide was 100 mg/lOO ml. One ml of each of 0.005N KI03, 0.016
KI, and 0.5N HCl were then addec to a total of 1 ml of
standard/sample and final volume (9 ml) made up with distilled
water. Absorbance was read at 615 nm in Beckman DEG
spectrophotometer.
Water and oil absorption Water and oil (Crisco vegetable cooking
oil, density = 0.8888
g/ml) absorption capacities of the purified starch and the
modified starches were deternined by the centrifugal method
(Beuchat, 1977). One gram 0: sample was mixed with 10 ml of
distilled water/oil (Sari-whirl, mixing control, fast) for 30 set,
allowed to
618-Volume 46 /1981)-JOURNAL OF FOOD SCIENCE
stand for 30 min at room temperature (21(Z), centrifuged at 5000
X G for 30 mm and the volume of the supernatant noted. Density of
distilled water was assumed to be 1 g/ml. Results were expressed on
a dry weight basis.
Pasting properties Starch gelatinization curves were obtained by
the method of
Sandstedt and Abbott (1964). Starch (2Og, dry weight basis) was
suspended in 350 ml of distilled water in a Waring Blendor. CMC
(Cellulose Gum 7 HP, Hercules Powder Co.. Wilminaton. Del.) was
added (3.6g) with gentle stirring over 30 set to this suspension,
blended for 1 min, and poured into the amylograph bowl. The blender
was then rinsed with 100 ml distilled water and the water was added
to the amylograph bowl. The temperature of this starch- CMC
suspension was then raised from 25C to 95C at a rate of 1.5C/min;
held at 95C for 15 min and then cooled uniformly to 50C (1.5C/min).
A blank curve for CMC was prepared similarly (with 3.6g of CMC
alone) and subtracted from the starchCMC curves.
Gelation The method of Coffman and Garcia (1977) was employed
with
slight modifications. Purified starch suspensions of
1,3,5,7,9,11, 13, 15, 17, and 20% (w/v) were prepared in 5 ml
distilled water and the test tubes were heated in a boiling water
bath for 1 hr followed by rapid cooling under running cold tap
water. The test tubes were further cooled for 2 hr at 4C. Least
gelatinization concentration was determined as that concentration
when the sample from the inverted test tube did not fall down or
slip.
Degree of substitution The degree of substitution (D-S.) for the
acetylated starch was
determined according to Wurzburg (1964). Starch (5g) was dis-
persed in 75% aqueous ethanol and warmed for 30 mm on a water bath
(5OC), cooled to room temperature (21C), and 25 ml of 1N NaOH
added. The stoppered flasks were then allowed to stand for 72 hr
with occasional shaking at room temperature (21C). Excess NaOH was
back titrated with 1N HCl. The flasks were al- lowed to stand at
room temperature (21C) for 2 hr and the titra-
Table 1-Physichochemical data on the Great Northern bean
starch
Yield Moisture Protein FatC Sample (%) Pd 1%) (%)
Crude starch 87.50a 3.07 4.86 0.34 Purified starch 1 8.23a 2.67
0.97 0.46 Acetylated starchd 92.00b 3.70 - - Oxidized starch 84.40b
4.12 - -
a On bean flour basis b On purified starch basis i Dry weight
basis
Degree of substitution (D.S.) = 0.40
Fig. 2-Light photomicrograph of Great North&m bean starch I1
75X).
-
GREATNORTHERN BEAN STARCH.. .
tion completed. A blank with pure starch was conducted concur-
rently and the D.S. calculated as follows:
% Acetyl =
(ml blank - ml sample) X normality of HCl X 0.43 X 100 Weight of
sample (g) dry basis
D.S. = 162 X % Acetyl 4300 - (42 X % Acetyl)
RESULTS & DISCUSSION
Composition and yield The data on composition and yield are
presented in
Table 1. The purity of the isolated starch was judged on the
basis of composition and microscopic observations. The yield of
Great Northern bean starch was 18.23% (on bean flour basis).
Naivikul and DAppolonia (1979) re- ported yields of 40.3, 38.3,
39.9,42.5, and 34.5% for navy bean, pinto bean, faba bean, lentil,
and mung bean starch respectively. Schoch and Maywald (1968)
obtained starch yields of 27, 38, and 37% from navy bean, lentil,
and mung bean seeds respectively. Lineback and Ke (1975) reported
37% starch yield from horsebean flour. The differences in yields
have been attributed to the methods opted for starch isolation
(Naivikul and DAppolonia, 1979). A yield of 18.23% in the present
investigation which was lower than those of navy and pinto beans,
both Phaseolus vulgaris species may be primarily due to the method
of isolation.
Granule size and microscopic appearance The shape, size, and
bifringence of starch granules are
often representative of the plant species and its maturity
(Manners, 1974). Several fiels were observed and measure- ments of
25 representative granule sizes were made. The range of granule
size was about 12 X 12 pm to 58 X 40 pm (length X width) which was
in close agreement to the ranges reported by Naivikul and
DAppolonia for navy and pinto bean starches (12-36 and 16-28 pm for
width, 1240 and 1640 pm for length, respectively, for navy and
pinto bean starch granules). The shape of Great Northern bean
starch granules was quite varient, ranging from small round to
large oval to irregular. Some granules were con- cave. Similar
observations have been reported by Line- back and Ke (1975) on
chick pea and horsebean starches, and by Lai and Varriano-Marston
(1979) on black bean starch.
Light microscopic observations (Fig. 2) of the starch granules
revealed the presence of hylum and lamellae. In general, the hylum
paralleled the longitudinal axis of the starch granule; however, in
case of spherical granules such as distinction could not be made.
Hylum was absent on some granules; however, lamellae were observed
in all the granules viewed. Hylum was found to possess different
shapes and varying lengths. Similar observations have been reported
on starch grains of Phaseolus species (Dhaliwal et al., 1964) and
lima beans (Salunkhe and Pollard, 1955a, b; Salunkhe, 1957).
Scanning electron photomicrographs are presented in Figure 3. As
can be seen from these photographs, starch granules appear to be
round, oval, and elliptical. The sur- faces appeared to be smooth.
The lamellae observed in the light microscopic view (Fig. 2) were
not evident in the scanning electron microscopic observations on
starch gran- ules. This may have been due to the dehydrated state
of starch granules in scanning electron microscopy samples versus
the hydrated state in light microscouv. Some starch granules
appeared to be do-nut shaped. -Hall and Sayre (1971), McEwen et al.
(1974), and Schoch and Maywald
(1968) have reported similar observations on legume starches.
The cell wall structure is shown in Figure 4. The starch granules
can be seen enclosed in the cell wall. The isolation treatments did
not remove completely all the cell walls and some did survive as
shown in the Figure 4.
Amylose content and starch hydrolysis The amylose content of
Great Northern bean starch was
Fig. 3-Scanning electron photomicrographs of Great Northern
I
bean starch: (Al 365X; (B1 730X; (Cl 1460X.
Volume 46 (1981bJOURNAL OF FOOD SCIENCE-619
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10.2% (on starcll basis) which was comparable (in order) to that
reported in Amsoy 71 soybeans (15-20%) by Wil- son et al. (1978).
Results of starch hydrolysis employing a-amylases are p resented in
Figures 5 and 6. Hog pancreatic a-amylase hydrolysed 8.2% starch
which was higher than that by cw-amzla:e from malt, in a 2-hr
period at room tem- perature (21 C). This difference may be due to
the differ- ence in the activities of enzymes used (11 mg maltose
and 3.3 mg maltose liberated per mg of enzyme in 1 min at 20C at pH
6.9 for hog and malt ol-amylase, respectively). The low degree of,
hydrolysis may be due to the starch nature (uncookell raw starch
was employed) and a relative- ly low temperature (21C) during
incubation.
Water and oil abr orption The water alld oil absorption data are
presented in
Table 2. Modifications did not improve both water and oil
absorption capac:ity of starch. The purified starch had oil and
water absorption capacity of about 2.9 g/g and 2.93 g/g,
respectively. Halbrook and Kurtzman (1975) have reported a water
uptake of about 3.0 g/g and about 3.0 g/g at 121C and 80C,
respectively. Our results of water absorption (2.92 g/g at 21C for
the purified starch) were comparable to .:heir observation of water
absorption at 8OC. The high water absorption at 21C observed in the
present investigation may have been due to the nature of
Fig. 4-Scanning eh&on photomicrograph of the cell wail,
1460X.
the starch and a possible contribution to water absorption by
the cell wall material(s) which was not removed com- pletely. Comer
and Fry (1978) have reported cold water absorption of the purified
pea starch to be 92-105%, and that the water uptake was a function
of temperature.
Pasting properties and gelation The amylograms are the plots for
the corrected viscosity
(Fig. 7). The data are summarized in Table 3. Peak heights were
not reported as the amylograms did not have distinct peaks. With
the exception of the oxidized starch, all other samples followed
similar patterns. The change in viscosity after holding for 15 min
at 95C was rather slow, except for oxidized starch in which case it
decreased sharply dur- ing the cooling cycle. The gelatinization
temperature range (65.5-68.5(Z) of the purified Great Northera bean
starch was comparable to those of faba bean (66 Cd and lentil (68C)
(Naivikul, 1977); garbanzo bean (65-71 C), smooth pea (65-69(Z),
red kidney bean (64-68OC), and mung bean (63-69C) (Biliaderis et
al., 1979); and black bean (63.8-76C) (Lai and Varriano-Marston,
1979) starches. The trend of the purified starch curve was
characteristic of restricted swelling type starches. The viscosity
behavior of the oxidized starch was characteristic of hypochlorite
oxidized starches which show a greater degree of fluidity.
Table 2-Water andoilabsorption by the Great Northern bean
starch
Sample
Purified starch Acetylated starch Oxidized starch
Water absorbed Oil absorbed Sk Sk
2.93 2.94 2.68 1.88 2.60 2.26
Table 3-Amylogram Data of the Great Northern bean starch
Sample
Crude starch Purified starch Acetylated starch Oxidized
starch
Gelatinization temp range
(C)
62.587.0 65.5-68.5 61.0-64.0 65.5-68.5
15 min
6
425 295 355
40
50C htb (BU)
325 445 475
Ii
a Viscosity of the corrected starch curveO(in Brabender Units)
at the end of 15 min period of holding at 95 C.
b Viscosity at 50C (In Brabender Units) during the cooling
cycle.
Fig. 5-Light photllmicrograph of a-amylase (hog pancreas) attack
on starch granule. (A) 0 hr; IBJ 2 hr. The magnification of B is
2.5 times that ofA. I
620-Volume 41; (198lkJOURNAL OF FOOD SCIENCE
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GREAT NORTHERN BEAN STARCH.. .
The tendency to set back on cooling is minimized in oxi- dized
starches due to the presence of functional groups that block the
association tendencies of the starch chains (Scallet and Sowell,
1967). The gelation studies indicated that purified starch could
yield stable gels at concentrations of 7% or above (w/v).
O-0 Hog d-Amylare
- . . . . . . . . M&d-Amyline
I I I I I a 1 I 30 45 60 75
TIME (min.) -*
90 105 120
Fig. 6-Starch hydrolysis by or-am ylases.
Fig. 7-Gelatinization curves (corrected for CMC) of Great
Northern bean starch.
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car- bohydrate composition of legume seeds: Horsebeans. peas, and
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Dhaliwal. AS.. Pollard. L.H.. and Salunkhe. D.K. 1964.
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Hall. D.M. and Sayre, J.G. 1971. A scanning electron microscope
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Hullinger. C.H. 1964. Hypochlorite oxidized starch. In Methods
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313. Academic Press, New York.
Lai, C.C. and Varrlano-Marston, E. 1979. Studies on the charac-
teristics of black bean starch. J. Food Sci. 44: 528.
Lineback. D.R. and Ke, C.H. 1975. Starches and low molecular
weight carbohydrates from chick pea and horsebean flours. Cereal
Chem. 52: 334.
Manners, D.J. 1974. The structure and metabolism of starch. In
Essays in Biochemistry, Vol. 10, p. 37. Ed. Campbell, P.N. and
Dickens, F.. Academic Press, New York.
McEwen. T.J., McDonald, B.E.. and Bushuk. W. 1974. Faba bean
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Unpublished report, The Fourth International Food Congress, Madrid,
Spain.
Naivikul, 0. 1977. The carbohydrates present in flour obtained
from various types of legumes. Ph.D. thesis, North Dakota State
University, Fargo, N.D.
Naivikul. 0. and DAppolonia. B.L. 1979. Carbohydrates of legume
flours compared with wheat flour. 2. Starch. Cereal Chem. 56:
24.
Salunkhe, D.K. 1957. Histological and histochemical changes in
gamma-irradiated lima beans, Phaseolus lunatus. Nature 179:
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Salunkhe. D.K. and Pollard, L.H. 1955a. A rapid and simple
method to determine the maturity and quality of lima beans. Food
Tech- nol. 9: 45.
Salunkhe, D.K. and Pollard, L.H. 195513. Further studies on
micro- scopic examination of starch grains in relation to maturity
of lima beans. Food Technol. 9: 521.
Sandstedt, R.M. and Abbott, R.C. 1964. A comparison of methods
for studying the course of starch gelatinization. Cereal Sci. Today
9: 13.
Sathe, S.K., Ponte, J.G. Jr., Rangnekar, P.D., and Salunkhe,
D.K. 1980. Effects of addition of Great Northern bean (Phaseolus
vul- garis L.) flour and protein concentrates on rheological
properties of dough and baking quality of bread. Cereal Cbem. (In
press).
Scallet, B.L. and Sowell, E.A. 1967. Production and use of hypo-
chlorite oxidized starches. In Starch Chemistry and Technology.
Industrial Aspects, Vol. 2, p. 243. Ed. Whistler, R.L. and
Paschall. E.F.. Academic Press, New York.
Schoch, T.J. 1964. Fatty substances in starch. Determination and
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MS received 6/26/80; revised E/16/80; accepted E/24/80.
Presented at the 40th Annual Meeting of the Institute of Food
Technologists, New Orleans, La., June E-11.1980.
Contribution No. 2578 from the Utah Agriculture Experiment
Station and a contribution of Western Regional Project W-150.
We thank Professor J.G. Ponte Jr. and Mr. P.D. Rangnekar. Dept.
of Grain Science & Industry, Kansas State Univ., Manhattan, KS
66506, for their help in viscoamylographic studies.
Volume 46 /1981)-JOURNAL OF FOOD SCIENCE-621